title: HLA-universal platelet transfusions prevent platelet refractoriness in a mouse model.
abstract: Refractoriness to platelet (PLT) transfusion caused by alloimmunization against HLA class I antigens constitutes a significant clinical problem. Thus, it would be desirable to have PLT units lacking HLA antigens on the cell surface. Previously, we showed that the generation of functional HLA class I-silenced (HLA-universal) PLTs from CD34(+) cells, using a short hairpin RNA (shRNA) to target β2-microglobulin (β2m) transcripts, is feasible. Here, we assessed the capacity of HLA-silenced PLTs to escape HLA antibody-mediated cytotoxicity in vitro and in vivo. Generation of megakaryocytes (MKs) and PLTs was performed by thrombopoietin-mediated differentiation of HLA-silenced CD34(+) cells within 10 days. Lymphocytotoxicity and antibody-dependent cellular cytotoxicity (ADCC) reporter assays using anti-HLA antibodies and a mouse model for PLT refractoriness were used to assess the immune-evasion capability of HLA-universal MKs and PLTs. To mimic PLT refractoriness in vivo, NOD/SCID/IL-2Rγc(-/-) mice were injected with specific anti-HLA antibodies followed by the infusion of 1 × 10(6) HLA-universal MKs. In vivo PLT generation was evaluated by flow cytometry using anti-CD42a and CD61 antibodies. Cells expressing a nonspecific shRNA were used as control. Lymphocytotoxicity and ADCC reporter assays showed that HLA silencing protects MKs against HLA antibody-mediated complement-dependent and cell-mediated cytotoxicity. In lymphocytotoxicity assays, 80-90% of HLA-expressing MKs but only 3% of HLA-silenced MKs were lysed. In the circulation of mice, HLA-expressing and HLA-silenced MKs showed PLT production in the absence of anti-HLA antibodies, with human PLT frequencies of up to 0.5% within the PLT population. However, in presence of anti-HLA antibodies HLA-expressing MKs were rapidly cleared from the circulation of mice, whereas HLA-silenced MKs escaped HLA antibody-mediated cytotoxicity and produced PLTs that were detectable up to 11 days. Our data show that HLA-silenced PLTs are efficiently protected against HLA antibody-mediated cytotoxicity and prevent PLT refractoriness in vivo. Provision of HLA-silenced PLTs may become an important component in the management of patients refractory to PLT transfusion.
authors.affiliation: 1 Institute for Transfusion Medicine, Hannover Medical School , 30625 Hannover, Germany .
authors.forename: Christiane
authors.initials: C
authors.lastname: Gras
authors.forename: Kai
authors.initials: K
authors.lastname: Schulze
authors.forename: Lilia
authors.initials: L
authors.lastname: Goudeva
authors.forename: Carlos A
authors.initials: CA
authors.lastname: Guzman
authors.forename: Rainer
authors.initials: R
authors.lastname: Blasczyk
authors.forename: Constança
authors.initials: C
authors.lastname: Figueiredo
mesh_terms: D000818:Animals; D018952:Antigens, CD34 / Q000235:genetics; D000954:Antigens, Surface / Q000235:genetics / Q000276:immunology*; D001792:Blood Platelets / Q000276:immunology*; D003602:Cytotoxicity, Immunologic; D004195:Disease Models, Animal; D020868:Gene Silencing; D015395:Histocompatibility Antigens Class I / Q000235:genetics / Q000276:immunology*; D006801:Humans; D051379:Mice; D017713:Platelet Transfusion / Q000009:adverse effects*; D034741:RNA, Small Interfering / Q000235:genetics
chemical_list: D018952:Antigens, CD34; D000954:Antigens, Surface; D015395:Histocompatibility Antigens Class I; D034741:RNA, Small Interfering